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Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦

View Article: PubMed Central - PubMed

ABSTRACT

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.

No MeSH data available.


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Protection of FraC from PK in the presence of liposomes.a, SDS-PAGE of the products obtained after the incubation of WT and 8-69OX FraC with DOPC vesicles (lanes 1–4) or with SM/DOPC (1:1) (lanes 5–8) in the absence and in the presence of PK. b, N-terminal sequence of FraC after digestion with PK. The circled numbers correspond to the lane in the SDS-PAGE. Residues highlighted in red were digested by PK. The first 16 residues of the recombinant WT protein expressed in E. coli are ADVAGAVIDGAGLGFD (54). c, location of the residues digested by PK (in red) are depicted in the three-dimensional structure of the monomer of FraC (Protein Data Bank code 3VWI).
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Figure 7: Protection of FraC from PK in the presence of liposomes.a, SDS-PAGE of the products obtained after the incubation of WT and 8-69OX FraC with DOPC vesicles (lanes 1–4) or with SM/DOPC (1:1) (lanes 5–8) in the absence and in the presence of PK. b, N-terminal sequence of FraC after digestion with PK. The circled numbers correspond to the lane in the SDS-PAGE. Residues highlighted in red were digested by PK. The first 16 residues of the recombinant WT protein expressed in E. coli are ADVAGAVIDGAGLGFD (54). c, location of the residues digested by PK (in red) are depicted in the three-dimensional structure of the monomer of FraC (Protein Data Bank code 3VWI).

Mentions: The incubation of PK with FraC in the presence of DOPC vesicles yields a fragment of smaller molecular weight than that of the untreated protein (shown in the 20-kDa region). In contrast, in the presence of SM/DOPC (1:1) vesicles, the bands of treated and untreated toxin display the same molecular mass (Fig. 7A). The mutein 8-69OX bound to membranes was also employed, because its N terminus remains exposed to the solvent constrained by the disulfide bond. As expected, 8-69OX was also susceptible to the proteolytic activity of PK in DOPC and SM/DOPC (1:1) vesicles. To determine the extent of the cleavage, the proteins were subjected to sequencing of their N-terminal regions. The sequencing data revealed that, in the presence of vesicles of DOPC, FraC WT and 8-69OX were cleaved at the N terminus by PK, rendering products in which the first 4 and the first 11 residues, respectively, were missing (Fig. 7, B and C). FraC bound to SM/DOPC (1:1) was not digested by PK as expected from the position of the band in the SDS-polyacrylamide gel, whereas 8-69OX was cleaved at the same position seen after incubation with vesicles of DOPC. These results demonstrate that the N terminus of FraC in DOPC vesicles (prepore configuration) is accessible to PK, i.e. this region is not embedded in the lipid bilayer.


Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins * ♦
Protection of FraC from PK in the presence of liposomes.a, SDS-PAGE of the products obtained after the incubation of WT and 8-69OX FraC with DOPC vesicles (lanes 1–4) or with SM/DOPC (1:1) (lanes 5–8) in the absence and in the presence of PK. b, N-terminal sequence of FraC after digestion with PK. The circled numbers correspond to the lane in the SDS-PAGE. Residues highlighted in red were digested by PK. The first 16 residues of the recombinant WT protein expressed in E. coli are ADVAGAVIDGAGLGFD (54). c, location of the residues digested by PK (in red) are depicted in the three-dimensional structure of the monomer of FraC (Protein Data Bank code 3VWI).
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Related In: Results  -  Collection

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Figure 7: Protection of FraC from PK in the presence of liposomes.a, SDS-PAGE of the products obtained after the incubation of WT and 8-69OX FraC with DOPC vesicles (lanes 1–4) or with SM/DOPC (1:1) (lanes 5–8) in the absence and in the presence of PK. b, N-terminal sequence of FraC after digestion with PK. The circled numbers correspond to the lane in the SDS-PAGE. Residues highlighted in red were digested by PK. The first 16 residues of the recombinant WT protein expressed in E. coli are ADVAGAVIDGAGLGFD (54). c, location of the residues digested by PK (in red) are depicted in the three-dimensional structure of the monomer of FraC (Protein Data Bank code 3VWI).
Mentions: The incubation of PK with FraC in the presence of DOPC vesicles yields a fragment of smaller molecular weight than that of the untreated protein (shown in the 20-kDa region). In contrast, in the presence of SM/DOPC (1:1) vesicles, the bands of treated and untreated toxin display the same molecular mass (Fig. 7A). The mutein 8-69OX bound to membranes was also employed, because its N terminus remains exposed to the solvent constrained by the disulfide bond. As expected, 8-69OX was also susceptible to the proteolytic activity of PK in DOPC and SM/DOPC (1:1) vesicles. To determine the extent of the cleavage, the proteins were subjected to sequencing of their N-terminal regions. The sequencing data revealed that, in the presence of vesicles of DOPC, FraC WT and 8-69OX were cleaved at the N terminus by PK, rendering products in which the first 4 and the first 11 residues, respectively, were missing (Fig. 7, B and C). FraC bound to SM/DOPC (1:1) was not digested by PK as expected from the position of the band in the SDS-polyacrylamide gel, whereas 8-69OX was cleaved at the same position seen after incubation with vesicles of DOPC. These results demonstrate that the N terminus of FraC in DOPC vesicles (prepore configuration) is accessible to PK, i.e. this region is not embedded in the lipid bilayer.

View Article: PubMed Central - PubMed

ABSTRACT

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of β-PFTs. However, in the class of α-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an α-PFT.

No MeSH data available.


Related in: MedlinePlus